Estrogens play essential roles in physiology and pathology of human development and reproduction. High-levels of the most active estrogen, 17beta-estradiol, have been linked to hormone-dependent breast cancer. Three enzymes primarily responsible for intracrine biosynthesis of nearly all of 17beta-estradiol in postmenopausal women and also in breast tumor cells are Cytochrome P450 Aromatase (P450arom), Type 1 17beta- Hydroxysteroid Dehydrogenase (17beta-HSD1) and Estrone/DHEA Sulfatase (ES). Although significant advances have been made in our laboratory towards understanding the structure-function relationship of 17beta-HSD1, three-dimensional structures of P450arom (55kDa) and ES (Subunit: 64kDa) are not yet available. The major objective of this proposed research is crystallization of two membrane-bound naturally-occurring, full-length, fully-active human enzymes P450arom and ES, in order to proceed with their high-resolution crystal structure analysis. No mammalian cytochrome P450 or steroid sulfatase has been crystallized to date. We have optimized the purification processes for both enzymes from microsomal fractions of human placenta and are able to routinely obtain milligram quantities of the highly purified, homogeneous enzymes suitable for crystallization. We have completed the initial biochemical characterization and substrate-specificity studies of purified ES. We have prepared an activity-suppressing monoclonal antibody to P450arom. Single microcrystals of P450arom, showing the characteristic red coloration and active enzyme, have been obtained. We have initiated cloning of the variable domain Fv of the anti-P450arom antibody. The plan is to use the Fv fragment in P450arom crystallization and crystal structure analysis of the P450arom-Fv complex. Affinity-optimized recombinant anti- P450arom Fv could have therapeutic as well as diagnostic applications. Most of the anti-breast cancer agents in use to-day, such as tamoxifen or its analogs, work by blocking the estrogen receptor and have been shown to have serious side-effects. The enzymes pose attractive alternative targets for lowering estrogen levels in breast tumor tissues. Simultaneous inhibition of two or all three enzymes could not only be one of the most effective treatments of breast cancer, but also provide means for its prevention in high-risk populations. The long-term objective is structure-based design of inhibitors with high specificities, but mutually exclusive affinities for their respective targets, and no affinity for the estrogen receptor.